Assay for the detection of paclitaxel resistant cells in human tumors

Abstract

Tubulin mutations commonly associated with resistance to paclitaxel are defined, and PCR allele-specific primers capable of detecting the mutations in DNA from tumor cells are described as well as method for treating paclitaxel-resistant cells in tumors. A simple, rapid, and cost effective means for detecting paclitaxel-resistant cells in tumor biopsies from patients receiving paclitaxel therapy is disclosed.

We claim:  
     
         1 . A purified polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, and anti-sense analogs thereof, where the sequence is characterized by conferring paclitaxel-like drugs resistant to cells in which it is expressed.  
     
     
         2 . The polynucleotide of  claim 1 , wherein the polynucleotide is a probe.  
     
     
         3 . The polynucleotide of  claim 2 , wherein the probe binds anti-sense nucleic acids including a mutation in a beta-tubulin encoding sequence corresponding to the sequence.  
     
     
         4 . An isolated tubulin amino acid sequence comprising an amino acid sequence having at least one mutation, the mutation selected from the group consisting of a mutation at position 210, a mutation at position 214, a mutation at position 215, a mutation at position 216, a mutation at position 217, a mutation at position 225, a mutation at position 228, a mutation at position 270, a mutation at position 273, a mutation at position 292, and a mutation at position 365 and any combination thereof.  
     
     
         5 . The amino acid sequence of  claim 4 , wherein the mutation is selected from the group consisting of SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, and SEQ ID NO. 45.  
     
     
         6 . A test kit useful for the detection of beta-tubulin mutations comprising a container containing at least one polynucleotide capable of hybridizing with a polynucleotide encoding at least one mutation at one of the following positions: 210, 214, 215, 216, 217, 225, 228, 270, 273, 292 and 365.  
     
     
         7 . A method to determine if a tumor cell is resistant to drugs which affect microtubule assembly, comprising the steps of: 
 (a) extracting a polynucleotide including an encoding region of beta-tubulin from a tumor cell;    (b) amplifying the polynucleotide; and    (c) determining if the polynucleotide includes an amino acid changing mutation at an amino acid site of beta-tubulin selected from the group consisting of 210, 214, 215, 216, 217, 225, 228, 270, 273, 292, 365 and mixtures or combinations thereof, where the mutation confers resistance to the drugs.    
     
     
         8 . The method of  claim 7 , wherein the amplifying is polymerase chain reaction amplifying.  
     
     
         9 . The method of  claim 7 , wherein the drugs are compositions selected from the group consisting of taxanes, epothilones, discodermolides and analogs thereof.  
     
     
         10 . The method of  claim 9 , wherein the taxanes are composition selected from the group consisting of paclitaxel, taxotere and analogues thereof.  
     
     
         11 . The method of  claim 7 , wherein the drugs stabilize microtubule assembly.  
     
     
         12 . The method of  claim 7 , further comprising the step of: 
 (d) administering a non-pactitaxel oncologic medication to the patient if the mutation is detected.    
     
     
         13 . A therapeutic method to treat cancer patients who are resistant to treatment comprising the steps of: 
 (a) isolating a tumor cell from a patient;    (b) extracting a polynucleotide encoding a region of beta-tubulin from the tumor cell;    (c) amplifying the polynucleotide;    (d) determining if the polynucleotide includes an amino acid changing mutation at an amino acid site of beta-tubulin selected from the group consisting of 210, 214, 215, 216, 217, 225, 228, 270, 273, 292, 365 and mixtures or combinations thereof, where the mutation confers resistance to drugs which stabilize microtubule assembly; and    (e) administering a non-pactitaxel oncologic medication to the patient if the mutation is detected.    
     
     
         14 . The method of  claim 13 , wherein the medication is an antimitotic drug that inhibits microtubule assembly.  
     
     
         15 . The method of  claim 14 , wherein the antimitotic drug is selected from the group consisting of vinblastine, vincristine and analogs thereof.  
     
     
         16 . A method to determine if a tumor cell is resistant to drugs, which affect microtubule assembly, comprising the steps of: 
 (a) extracting a beta-tubulin polypeptide from a tumor cell; and    (b) determining if the polypeptide includes an amino acid substitution at amino acid sites selected from the group consisting of 210, 214, 215, 216, 217, 225, 228, 270, 273, 292, 365 and mixtures or combinations thereof, where the substitution confers resistance to the drugs.    
     
     
         17 . The method of  claim 16 , wherein the drugs are compositions selected from the group consisting of taxanes, epothilones, discodermolides and analogs thereof.  
     
     
         18 . The method of  claim 17 , wherein the taxanes are composition selected from the group consisting of paclitaxel, taxotere and analogues thereof.  
     
     
         19 . The method of  claim 16 , wherein the drugs stabilize microtubule assembly.  
     
     
         20 . A method of screening a patient comprising the steps of: 
 (a) obtaining a sample from the patient;    (b) determining if one or more amino acid substitutions in a beta-tubulin polypeptide from the patient sample are present; and    (c) administering a microtubule assembly stabilizing drug to the patient if no amino acid substitution in the beta-tubulin polypeptide is present; or    (d) administering a microtubule assembly inhibiting drug to the patient if one or more claims of the substitutions is present.    
     
     
         21 . The method of claim  22 , wherein the substitutions are selected from the group consisting of amino acid positions 210, 214, 215, 216, 217, 225, 228, 270, 273, 292, 365, and any combination thereof.